Novel proteins

ABSTRACT

The present invention relates to human diacylglycerol kinase proteins (hDAGK) and particularly to human diacylglycerol kinase β (hDAGKβ) protein, and to related nucleotide sequences, expression vectors, cell lines, antibodies, screening methods, compounds, methods of production and methods of treatment, as well as other related aspects.

FIELD OF THE INVENTION

[0001] The present invention relates to human diacylglycerol kinaseproteins (hDAGK) and particularly to human diacylglycerol kinase β(hDAGKβ) protein, and to related nucleotide sequences, expressionvectors, cell lines, antibodies, screening methods, compounds, methodsof production and methods of treatment, as well as other relatedaspects.

BACKGROUND OF THE INVENTION

[0002] Diacylglycerol kinases (DAGKs) are a family of enzymes thatconvert diacylglycerol (DAG) to phosphatidic acid and are thereforeknown to attenuate DAG-dependent protein kinase C activation (PCK) (1).

[0003] Five types of DAGKs have been described. Type I DAGKs containsfour conserved regions, the N-terminal region (C1), two sets of EF-handmotifs (C2), two cysteine-rich zinc finger like structures (C3) and theC-terminal region (C4). Type II isoenzymes contain a pleckstrin homology(PH) domain at the N-terminus. Type III contains only the zinc finger(C3) and the catalytic region (C4). Type IV contains four ankyrinrepeats near the carboxyl terminus. Type V contains three instead of twozinc finger structures, a proline-rich region and a PH domain with anoverlapping Ras-associating (RA) domain. All share two domains, the C2and the C3 (2).

[0004] A 90 kDa DAGKβ (3) belonging to type I was found by screening arat brain cDNA library using fragments of rat a DAGK cDNA under lowstringency conditions. The cDNA clone obtained was completely sequenced.The rat DAGKβ cDNA has an open reading frame of 5927 nucleotides andencodes for a protein of 801 amino acids with a predicted relativemolecular mass of 90,000. Analysis of the amino acid sequence identified2 EF-hand motifs (aa 152-180 and 197-225), of two cysteine-richzinc-finger-like sequences (aa 257-292 and 319-356), and putativeATP-binding sites (aa 266-294 and 533-560).

[0005] Brain expression has been described for the known DAGK isoforms(2), which include DAGKα, DAGKη, DAGKζ and DAGKθ. A particulardistribution restricted to specific regions of the central nervoussystem (CNS) was described for the DAGKβ form, originally identified inthe rat (90 Da DAGKβ). The rat beta form is predominantly localised inneurons of the caudate-putamen, the accubens nucleus and the olfactorytubercle. Such brain regions are among the main CNS dopaminergic,serotonergic, acetylcholinergic and glutamatergic terminal fields (4).

[0006] It has also been demonstrated that some metabotropic dopamine,serotonin, glutamate, acetylcholine and several peptide receptors arecoupled with the phosphoinositide signal transduction system (5).Lithium is known as one of the most effective therapies for bipolardisorders. Although the biological mechanisms of the mood stabilisingproperties of lithium are not well understood, experimental evidenceindicates that lithium modulates the phosphoinositide signaltransduction system (6) by inhibiting the phosphatase that liberatesinositol from inositol phosphate (IP), and by modifying the activity ofthe phospholipase C (PLC)-dependent signalling pathways, including thelevels of the second messenger diacylglycerol (DAG) that activatesprotein kinase C (PKC).

[0007] With this background in mind, the present inventors havedetermined that the DAGKs, and particularly the human ortholog of rat 90kDa DAGKβ (hDAGKβ) and variants thereof will provide targets for thedevelopment of novel mood stabilising agents and therapeutic agents fortreatment of other disorders.

[0008] Clearly there is a need for proteins and related nucleotidesequences that may be used to screen for mood stabilising agents andwhich may also play a role in preventing, ameliorating or correctingmood disorders and dysfunction and other neurological diseases.

[0009] Accordingly, it is an object of the present invention to identifytargets for screening of novel therapeutic agents. It is another objectof the invention to locate and characterise human DAGKβ and variantsthereof. Other objects of the present invention will become apparentfrom the following detailed description thereof.

SUMMARY OF THE INVENTION

[0010] According to one embodiment of the present invention there isprovided an isolated human diacylglycerol kinase β (hDAGKβ) protein or avariant thereof. In a particularly preferred embodiment of thisinvention the human diacylglycerol kinase β (hDAGKβ) has the amino acidsequence set out in Seq ID No 1 or Seq ID No 4. The hDAGKβ proteinhaving the amino acid sequence set out in Seq ID No 4 is referred to asSV-hDAGKβ protein.

[0011] According to one embodiment of the present invention there isprovided a nucleotide sequence encoding a human diacylglycerol kinase β(hDAGKβ) protein or a variant thereof, or a nucleotide sequence that iscomplementary thereto. In a particular preferred embodiment of theinvention the polynucleotide comprises the sequence set out in Seq ID No3 or Seq ID No 6. The hDAGKβ protein having the polinucletide sequenceset out in Seq ID No 6 is referred to as SV-hDAGKβ protein.

[0012] In accordance with another aspect of the invention there isprovided an expression vector comprising a nucleic acid sequence asreferred to above which is capable of expressing a hDAGKβ protein.

[0013] According to a further aspect of the invention there is provideda stable cell line comprising an expression vector as referred to above.Preferably the cell line is a modified HEK293T, CHO, HeLa, Sf9 or COScell line.

[0014] According to yet a further aspect of the invention there isprovided an antibody specific for a hDAGKβ protein.

[0015] According to still another aspect of the invention there isprovided a method for identification of a compound that exhibits DAGKmodulating activity, comprising contacting a DAGK protein with a testcompound and detecting modulation of enzyme activity or detecting enzymeinactivity. Preferably the DAGK is hDAGKβ or a variant thereof.

[0016] According to another aspect of the invention there is provided acompound which modulates hDAGK activity, identifiable by the methodreferred to above. Preferred compounds according to the presentinvention are those that modulate hDAGKβ activity or a variant thereof.

[0017] According to another aspect of the invention there is provided acompound that modulates hDAGK activity. Preferred compounds according tothe present invention are those that modulate hDAGKβ activity or avariant thereof.

[0018] According to a further aspect of the invention there is provideda method of treatment or prophylaxis of a disorder that is responsive tomodulation of hDAGK activity in a human patient, which comprisesadministering to said patient an effective amount of a compound asreferred to above. Conveniently the hDAGK is hDAGKβ or a variantthereof. Preferably the disorder is a mood disorder, epilepsy, aneurodegenerative disorder, anxiety, schizophrenia, migraine, drugdependence, stroke, Alzheimer's dementia or Parkinson's disease.

[0019] According to a further aspect of the invention there is provideda method of treatment or prophylaxis of a disorder that is responsive tomodulation of hDAGK activity in a human patient which comprisesadministering to said patient an effective amount of a modulator ofhDAGK activity. Conveniently the hDAGK is hDAGKβ or a variant thereof.Preferably the disorder is a mood disorder, epilepsy, aneurodegenerative disorder, anxiety, schizophrenia, migraine, drugdependence, stroke, Alzheimer's dementia or Parkinson's disease.

[0020] According to another aspect of the invention there is provideduse of a compound as referred to above in a method of formulating amedicament for treatment or prophylaxis of a disorder that is responsiveto modulation of hDAGK activity in a human patient. Conveniently thehDAGK is hDAGKβ or a variant thereof. Preferably the disorder is a mooddisorder, epilepsy, a neurodegenerative disorder, anxiety,schizophrenia, migraine, drug dependence, stroke, Alzheimer's dementiaor Parkinson's disease.

[0021] According to another aspect of the invention there is provideduse of a modulator of hDAGK activity in a method of formulating amedicament for treatment or prophylaxis of a disorder that is responsiveto modulation of hDAGK activity in a human patient. Conveniently thehDAGK is hDAGKβ or a variant thereof. Preferably the disorder is a mooddisorder, epilepsy, a neurodegenerative disorder, anxiety,schizophrenia, migraine, drug dependence, stroke, Alzheimer's dementiaor Parkinson's disease.

[0022] According to another aspect of the invention there is provided amethod of producing a hDAGKβ protein or a variant thereof comprisingintroducing into an appropriate cell line a suitable vector comprising anucleotide sequence encoding for a hDAGKβ protein or a variant thereof,under conditions suitable for obtaining expression of the hDAGKβ proteinor variant.

[0023] Seq ID No 3 shows the complete nucleotide sequence of the humanDAGKβ. Seq ID No 5 shows the complete nucleotide sequence of theSV-hDAGKβ.

[0024] Seq ID No 1 shows the nucleotide and encoded amino acid sequenceof the human DAGKβ sequence. Seq ID No 4 shows the nucleotide andencoded amino acid sequence of the SV-hDAGKβ.

[0025] Seq ID No 7 shows pairwise alignment of hDAGKβ and SV-hDAGKβfull-length amino acids sequences with rat homologue.

[0026] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

[0027]FIG. 1 shows a reverse transcriptase polymerase chain reaction(RT-PCR) of human adult and human foetal brain (polyA+RNA).

[0028]FIG. 2 shows the position of the alternatively spliced exons thatgenerate a family of hDAGKβ transcripts. The alternatively spliced exonsare illustrated as white boxes. The dashed box identifies the SV-hDAGKβtranscripts. Panel A: hDAGKβ transcripts generated through alternativesplicing. Panel B: SV-hDAGKβ transcripts generated through alternativesplicing.

[0029]FIG. 3 shows RT-PCR analysis of the expression of hDAGKβ andSV-hDAGKβ in human tissue cDNAs. PCR reactions were carried outseparately for each primer pair (hDAGKβ, SV-hDAGKβ and β-actin control)on the indicated tissue cDNAs, and reactions corresponding to the sametissue template were loaded in the same well.

[0030]FIG. 4 shows RT-PCR analysis of hDAGKβ and SV-hDAGKβ expression innormal and Alzheimer cerebellum. β-actin was used as a control of RNAlevels in the samples.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Throughout the present specification and the accompanying claimsthe words “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows.

[0032] As referred to above, the present invention relates to humandiacylglycerol kinase β (hDAGKβ) protein, sequence information for whichis provided in Seq ID No 1 or Seq ID No 4. In the context of thisinvention the term “isolated” is intended to convey that the protein isnot in its native state, insofar as it has been purified at least tosome extent or has been synthetically produced, for example byrecombinant methods. The term “isolated” therefore includes thepossibility of the protein being in combination with other biological ornon-biological material, such as cells, suspensions of cells or cellfragments, proteins, peptides, organic or inorganic solvents, or othermaterials where appropriate, but excludes the situation where theprotein is in a state as found in nature.

[0033] Routine methods can be employed to purify and/or synthesise theproteins according to the invention. Such methods are well understood bypersons skilled in the art, and include techniques such as thosedisclosed in Sambrook et al. (7), the disclosure of which is includedherein in its entirety by way of reference.

[0034] By the term “variant” what is meant throughout the specificationand claims is that other peptides or proteins which retain the sameessential character of the diacylglycerol kinase for which sequenceinformation is provided, are also intended to be included within thescope of the invention. For example, other peptides or proteins withgreater than about 80%, preferably at least 90% and particularlypreferably at least 95% homology with the sequences provided areconsidered as variants of the enzymes. Such variants may include thedeletion, modification or addition of single amino acids or groups ofamino acids within the protein sequence, as long as the peptidemaintains the biological functionality of a hDAGKβ. The rat DAGKβprotein is of course excluded from the definition of “variant”.

[0035] Human DAGKβ is expressed in human brain and has the highest (˜95%identity) sequence homology with the rat DAGKβ. Therefore, hDAGKβ islikely to be the human orthologue of rat DAGKβ. Seq ID No 1 reveals a3926 bp (base pair) open reading frame which encodes an 804 amino acidprotein. This deduced protein sequence is ˜95% identical to the ratDAGKβ and shares many of its characteristics and all the domains.

[0036] The invention also includes nucleotide sequences identified asSeq ID No 1 or Seq ID No 4 that encode for hDAGKβ protein or variantsthereof as well as nucleotide sequences that are complementary thereto.Preferably the nucleotide sequence is a DNA sequence and mostpreferably, a cDNA sequence. Such nucleotides can be isolated orsynthesised according to methods well know in Sambrook et al. (7), thedisclosure of which is included herein in its entirety by way ofreference.

[0037] The present invention also includes expression vectors thatcomprise nucleotide sequences encoding for the hDAGKβ protein orvariants thereof. A further aspect of the invention relates to anexpression vector comprising nucleotide sequences encoding for hDAGKβprotein or variants thereof. Such expression vectors are routinelyconstructed in the art of molecular biology and may for example involvethe use of plasmid DNA and appropriate initiators, promoters, enhancersand other elements, such as for example polyadenylation signals whichmay be necessary, and which are positioned in the correct orientation,in order to allow for protein expression. Suitable vectors would beapparent to persons skilled in the art. By way of further example inthis regard we refer to (7), the disclosure of which is included hereinin its entirety.

[0038] The invention also includes cell lines that have been modified toexpress the novel protein and variants thereof. Such cell lines includetransient, or preferably stable higher eukaryotic cell lines, such asmammalian cells or insect cells, lower eukaryotic cells, such as yeastor prokaryotic cells such as bacterial cells. Particular examples ofcells that have been modified by insertion of vectors encoding for theproteins according to the invention include the mammalian HEK293T, CHO,HeLa, Sf9 and COS cells.

[0039] It is also possible for the protein and variants thereof of theinvention to be transiently expressed in a cell line, such as forexample in a baculovirus expression or in an E. coli system. Suchsystems, which are adapted to express the proteins according to theinvention, are also included within the scope of the present invention.

[0040] According to another aspect, the present invention relates toantibodies (either polyclonal or preferably monoclonal antibodies) whichhave been raised by standard techniques and are specific for the proteinor variants thereof according to the invention. Such antibodies couldfor example, be useful in purification, isolation or screening involvingimmuno-precipitation techniques and may be used as tools to furtherelucidate protein function, or indeed as therapeutic agents in their ownright. Antibodies may also be raised against specific epitopes of theproteins according to the invention.

[0041] An important aspect of the present invention is the use of hDAGKproteins in screening methods designed to identify compounds which actas enzyme ligands and which may be useful as modulators of enzymaticactivity. In general terms, such screening methods will involvecontacting the hDAGK protein concerned, which may be any known or as yetunrecognised hDAGK protein or variant thereof, preferably hDAGKβ, with atest compound and then detecting modulation of the enzymatic activity,or indeed detecting enzyme inactivity, which results.

[0042] The present invention also includes within its scope thosecompounds, which are identified as possessing useful hDAGK modulationactivity. Such activity can be determined by the screening methodsreferred to above. The screening methods comprehended by the inventionare generally well known to those skilled in the art. An example of suchan approach is provided in the experimental section of thisspecification.

[0043] Another aspect of the present invention is the use of compoundswhich have been identified by screening techniques referred to above, orother compounds found to exhibit hDAGK modulating activity, in thetreatment or prophylaxis of disorders that are responsive to modulationof a hDAGK activity, particularly hDAGKβ activity, in a human patient.By the term “modulation” what is meant is that there will be eitheragonism or antagonism of the enzymatic activity, which results fromligand binding of the compound at the catalytic or regulatory sites ofthe hDAGK protein. These proteins have been implicated in disorders ofthe central nervous system (CNS), and therefore, modulation of hDAGKenzymatic activity in these tissues will result in a positivetherapeutic outcome in relation to such disorders.

[0044] In particular, the compounds which will be identified using thescreening techniques according to the invention will have utility fortreatment and/or prophylaxis of disorders such as mood disorders,epilepsy, anxiety, schizophrenia, drug dependence, neurodegenerativedisorders. Some specific examples of disorders which may be treated orprevented by administration of compounds identified in the screeningtechniques according to the present invention are unipolar and bipolardepression, stroke, Alzheimer's dementia, Parkinson's disease, smokingcessation, and ethanol, nicotine, cocaine and heroine abuse. It is to beunderstood, however, that the mention of such disorders is by way ofexample only, and is not intended to be limiting on the scope of theinvention.

[0045] The compounds which are identified according to the screeningmethods outlined above may be formulated with standard pharmaceuticallyacceptable carriers and/or excipients, as is routine in thepharmaceutical art, and as is fully described in Remmington'sPharmaceutical Sciences, Mack Publishing Company, Eastern Pennsylvania,17th Ed, 1985; the disclosure of which is included herein in itsentirety by way of reference.

[0046] The compounds may be administered via enteral or parenteralroutes such as via oral, buccal, anal, pulmonary, intravenous,intraarterial, intramuscular, intraperitoneal, topical or otherappropriate administration routes.

[0047] It will further be appreciated that the amount of a compound ofthe invention required for use in treatment will vary with the nature ofthe condition being treated, the route of administration and the age,sex, weight and general condition of the patient, and will ultimately beat the discretion of the attendant physician. In general, however, dosesemployed for adult human treatment will typically be in the range ofbetween about 2 mg to about 800 mg per day.

[0048] The present invention will be further explained, by way ofexample, in the following experimental section.

[0049] Experimental

[0050] Identification of Human Genomic Sequences Corresponding toBipolar Patients' ESTs Obtained by in Silico Analysis:

[0051] An extensive search using keywords in both public (GenBank) andprivate (Incyte) databases resulted in some ESTs annotated as specificfor bipolar diseases. Most of GenBank ESTs referred to the StanleyNeurovirology Laboratory (John Hopkins School of Medicine, Baltimore),where they were obtained by subtractive hybridisation of frontal cortexRNA from individuals with bipolar disorder and individuals withoutpsychiatric diseases as controls.

[0052] A first comparison against dbEST (8) using the Blastn (9)alignment program revealed that most of the bipolar specific ESTs do notcorrespond to any sequence of known function and no other overlappingESTs can be found to create contigs and enlarge the sequences.Therefore, in a preliminary analysis only those presenting enoughinformation to proceed with an in silico work were considered, inparticular those ESTs that showed 100% identity with a genomic sequence.The genomic sequence identified for the EST-S4 (GenBank acc. n.AF019352) is an unannotated 172-Kb length sequence (GenBank acc. n.AC005039) consisting of 2 contigs, for which the order is not known,interrupted by an N bases gap.

[0053] A detailed analysis was performed on the whole uncharacterisedregion with the aim of extracting all the high complexity sub-regionsthat usually contain coding sequences. The low complexity and highlyredundant regions found in portions of the sequence were isolated usingthe SEG (segment sequences by local complexity) tool (10, 11,) andcompared against all the available sequence databases to exclude anypossible translation. As expected, these low complexity regionscorresponded mainly to different families of ALU sequences thereforewere masked in the subsequent gene prediction approach.

[0054] The EST-S4 Belongs to a Novel Human Gene Orthologue to the RatDAGKβ Gene

[0055] The Blastx alignment program was used to compare the 4 Kb genomicregion surrounding the EST-S4 region with the amino acid sequences ofSwissProt and TREMBL databases. Only some local similarities with a highstatistical significance with the rat DAGKβ gene were evidenced andsuggest an authentic relationship. To verify if this result wasconsistent with a possible gene construct an in silico exon trappingmethod (12) was applied on the AC005039 sequence. GeneMark (13), Xpound(12) and GRAIL (14) exon prediction tools were used to locate thepotential coding regions within AC005039. Three exons (a, b and c) werefound to be consistent with the same ORF and to correspond to exons 22,23 and 24 of the rat DAGKβ gene. The region was 32 Kb wide and containedthe EST-S4 sequence.

[0056] Searching the more recent set of public domain nucleotidesequences (New GenBank updates), a partial mRNA sequence of 3742 bp froma human adult brain (KIAA0718, Acc. No. AB018261) was found to partiallyoverlap the 4 kb genomic sequence (100% identity with the three exons sofar identified). The predicted protein sequence (defined in GenPept asKIAA0718 protein) was limited to 742 aa with a N-terminal truncationdescribed. Homology search in protein databases indicates a highsimilarity with rat DAGKβ.

[0057] Chromosomal Localisation of the Putative hDAGKβ

[0058] The genomic sequence AC005039 is annotated as an unfinishedsequence mapped on chromosome 7. An “In silico” STS (Sequence TaggedSite) content analysis (15) was performed on the sequence, and the 3STSs (sWSS2950, D7S2174 and sWSS2190) found in the sequence confirmedthat the hDAGKβ is localised on 7p21.

[0059] In Silico Cloning of Full-Length of the hDAGKβ mRNA

[0060] Comparing (tBlastn) the first two exons of the rat proteinagainst the GenBank HTG (high throughput genomic) sequences database,two further overlapping genomic sequences (100% identity) wereidentified (GenBank acc. n. H_GS120K9 and AC006045). These sequences donot overlap with AC005039 but both contain four STSs (sWSS3226, sWSS822,sWSS2758, and sWSS2091) and belong to the same YAC clone (CEPH791G01)where AC005039 is located. These data indicate that the exons identifiedbelong to the hDAGKβ gene and provide the information missing in theKIAA0718 structure to complete the full length sequence of the predictedprotein

[0061] Identification and Characterisation of Human DAGKβ Variants incDNA Libraries.

[0062] Three DNA probes were used to screen a human foetal brain cDNAlibrary (cat. n. 936206 Stratagene, La Jolla, Calif.). The 246 bpfragment was obtained by reverse transcriptase polymerase chain reaction(RT-PCR) (16) with the oligo pairs previously described (see aboveparagraph), and used for radioactive hybridisation. The two primer pairsDAGKIAAfor/DAGKIAArev (5′ TGAAGACATTCCTGGAAGCC, 5′GACTGTGTACTTGCAGAAGG), and5hDAGKfor/5hDAGKrev (5′CCATGACAAACCAGGAAAAATGG, 5′ GATTATACTTTGCAAGCACACC) were used to obtain2 RT-PCR products of 647 bp and of 151 bp respectively from foetal brainpolyA+ RNA (Clontech) (FIG. 1, panel B).

[0063] The PCR conditions included an initial hot-start at 94° C. for 2minutes, followed by 35 cycles at 94° C. for 1 minute, 56° C.(DAGKIAAfor/DAGKIAArev) or 58° C. (5hDAGKfor/5hDAGKrev) for 1 minute,72° C. for 1 minute and terminated by 7 minutes at 72° C. The resultingPCR amplicons were separated on a 2% agarose gel and used forradioactive hybridisation.

[0064] To fully sequence the cDNA clones isolated, the library insertswere first subcloned in pBluescript KS vector (Stratagene, LaJolla,Calif.). After transformation, colonies were screened by hybridisationwith the previously described probes. Positive colonies were subjectedto direct sequencing (17) using the T3 and T7 primers. The DNA sequencesobtained were assembled using the GCG package, translated and alignedwith the rat DAGKβ gene using CLUSTAL (18). Two positive clones wereisolated using a human DAGKβ-specific probe covering sequences fromposition 1524 to position 2360 and their sequence covered by sequencingon both DNA strands. The clones contained the last three exons and partof the 3′ untranslated region (UTR) of the hDAGKβ sequence.

[0065] The tissue distribution of hDAGKβ gene was established byradioactive hybridisation on multi-Tissue northern blots (Clontech)according to the manufacturer's recommendations. The probes wereobtained by RT-PCR amplification of different portions of the codingregion of hDAGKβ including the 3′ splice variant specific probe.

[0066] Identification, Characterisation and Cloning of the 3′ end ofSV-DAGKβ Splice Variant.

[0067] The GCG package (Wisconsin Package Version 9.0, Genetics ComputerGroup (GCG), Madison, Wis.) was used to align the human genomic sequencesurrounding the EST-S4 with the rat 90 kDa DAGKβ mRNA. The EST-S4 islocated in the 30 Kb intron between exons b and c. It overlaps with theAC005039 genomic sequence 91 bp downstream the last amino acid of exonb. The genomic sequence contains two in frame stop codons and a possiblepolyadenylation signal is present on the EST sequence.

[0068] In order to verify if EST-S4 identifies a splice variant of thehuman DAGKβ gene, a reverse transcriptase polymerase chain reaction(RT-PCR) using the following primer pair: DAGK7for (5′TGCCMTGCAAATTGATGGG) and DAGK7rev (5′ AGCTAAATCATTGCCMGGG) that spanexon b and EST-S4 was performed. The PCR conditions included an initialhot-start at 94° C. for 5 minutes, followed by 35 cycles at 94° C. for 1minute, 56° C. for 1 minute and 72° C. for 1 minute and terminated by 5minutes at 72° C. The resulting PCR amplicon was separated on a 2%agarose gel and shown to be of 246 bp.

[0069] The transcript was successfully amplified in both human adult andfoetal brain polyA+ RNA (Clontech) RT-PCR reactions. As a control thehuman genomic DNA was also amplified, yielding a fragment of the samelength. The results indicate that the EST-S4 might correspond to the3′-UTR of a new splice variant (FIG. 1, panel A). Thus the shortest formof hDAGKβ protein encoded by the splice variant transcripts is hereindesignated as SV-hDAGKβ.

[0070] In order to further confirm the existence of an alternativesplicing event giving rise to a 3′ splice variant hDAGKβ isoform a 3′rapid amplification of cDNA ends (RACE) strategy (19) was applied tophysically identify and clone the relevant transcript portion in theform of cDNA. Briefly, human foetal brain polyadenylated RNA was reversetranscribed using the anchor oligonucleotideCCAGTGAGCAGAGTGACGAGGACTCGAGCTCMGC(T)₁₇ as a primer for first strandcDNA synthesis. The resulting cDNA was employed as a template for twonested rounds of PCR employing anchor-specific and gene-specific primers(first round RACE: CCAGTGAGCAGAGTGACG and TCAGAGCCACTACATTTAGGT; secondround RACE: GAGGACTCGAGCTCAAGC and AGGTTGTAGACATTATATACC). PCRconditions for both rounds were: 94° C. for 3 minutes (hot start); 25cycles of (94° C. for 30″, 56° C. for 30″, 72° C. for 30″) followed by a72° C. for 10′ step. Two RACE products, of 200 bp and 750 bprespectively, were obtained and cloned into appropriate E. coli plasmidcloning vectors. Double-pass sequencing confirmed the identity of thetwo products as two alternatively spliced transcripts bearing the 3′ endof the human DAGKβ splice variant coding sequence (with two predictedin-frame translational stop codons at position 2320 and 2365) and ca.100 bp or ca. 650 bp (owing to two alternatively used polyadenylationsignals) of 3′ untranslated sequence (UTR). Thus hDAGKβ splice varianttranscripts run out of the penultimate coding exon into the last intronof the DAGKβ locus, terminating at two alternatively usedpolyadenylation signals. The predicted protein encoded by the splicevariant transcripts (Seq ID No 4) lacks the last 30 amino acids presentin the longest (direct human orthologue of the rat DAGKβ) variant.

[0071] Physical Cloning of the Full Length Sequence of Human hDAGKβ andSV-hDAGKβ cDNAs

[0072] In order to clone the full length cDNA sequence encoding the twoDAGKβ variants, the sequence information derived from the in silico andcloning analyses was employed to design PCR primers comprising theinitiation codon of the protein (common to both variants) and thesequence immediately 3′ of the predicted translational stop codons ofeach variant. These primers were employed in two successive rounds ofnested long-range PCR (LR-PCR) employing a proofreading thermostablepolymerase (XL-PCR kit, Perkin Elmer, Calif.) according tomanufacturer's instructions. First round PCR conditions were as follows:94° C. for 3 minutes (hot start); 35 cycles of (94° C. for 30″, 55° C.for 30″, 72° C. for 5′) followed by a 72° C. for 20′ step. Second roundPCR was carried out on a 1 μl aliquot of a 1:10 dilution of first roundPCR using the same conditions as for first round PCR, except thatcycling was for 25 cycles. Primers were as detailed in Table 1.

[0073] The resulting PCR-amplified products (ca. 2.4 kb) were clonedinto appropriate plasmid cloning vectors and subjected to double passsequence analysis. This exercise confirmed the cDNA sequence predictedby the in silico analysis and extended it by revealing the presence ofthree alternatively spliced exons within hDAGKβ and SV-hDAGKβtranscripts and derived cDNAs (FIG. 5).

[0074] Alternative Transcript Splicing Generates Several Isoforms of thehDAGKβ and SV-hDAGKβ

[0075] Sequence analysis of the cloned full length cDNAs revealed thepresence of three alternatively spliced exons (encoding amino acidsequences of 7, 12 and 25 residues respectively) in addition to thepreviously characterized alternative splicing event leading to thegeneration of SV-hDAGKβ transcripts (FIG. 2). Thus at least 8 hDAGKβisoforms and 8 SV-hDAGKβ isoforms are predicted.

[0076] Expression of hDAGKβ and SV-hDAGKβ Transcripts in Adult HumanTissues

[0077] The specificity of hDAGKβ and SV-hDAGKβ expression in adult humantissues was investigated by RT-PCR. Polyadenylated RNA for a variety ofhuman tissues was obtained from a commercial source (Clontech, CA). Thismaterial was converted to cDNA and a set of PCR primers capable ofselectively amplifying either the hDAGKβ or the SV isoform were employedin an RT-PCR study. A set of β-actin specific primers were employed tocontrol the efficacy of the RT-PCR process. Table 1 provides the detailsof the primers. PCR conditions were: 94° C. for 3 minutes (hot start);40 cycles of (94° C. for 30″, 56° C. for 30″, 72° C. for 30″) followedby a 72° C. for 10′ step. The results (FIG. 3) indicate that both hDAGKβand SV-hDAGKβ are coordinately expressed in all tissues of neuronalorigin (brain regions and spinal cord). Non-neuronal tissues (with theexception of the uterus) do not express significant levels of hDAGKβtranscripts.

Table 1

[0078] PCR primers used in the present study. A. PCR primers employedfor the amplification of full length hDAGKβ cDNAs. The initiation(start) codon is underlined. B. PCR primers for the analysis ofexpression of h DAGKβ and SV-hDAGKβ isoforms. Sizes of expected productsare indicated. A DAGKβ transcript First round Second round hDAGKβCACCACCCATGACAAACCAGG ATGACAAACCAGGAAAAATGG And andTCTAAGAGTGAAACAACACAC AGGATTATTCCTTGCTTCGG SV-hDAGKβCACCACCATGACAAACCAGG ATGACAAACCAGGAAAAATGG And and AGCTAAATCATTGCCAAGGTCTACAACCTAAATGTAGTGG B DAGKβ transcript Primers amplified product (bp)hDAGKβ TGCCAATGCAAATTGA 153 And AGGATTATTCCTTGCTTCGG SV-hDAGKβTGCCAATGCAAATTGA 246 And AGCTAAATCATTGCCAAG β-actinTGAACCCTAAGGCCAACCGTG 400 And GCTCATAGCTCTTCTCCAGGG

[0079] Expression of hDAGKβ Transcripts in Normal vs NeuropathologicalConditions

[0080] The association between the hDAGKβ and the disorders arising fromabnormal expression/activity of hDAGKβ protein and variants thereof canbe illustrated by the following experiments.

[0081] The expression of hDAGKβ transcripts in normal and pathologicalconditions of relevance to the present patent was initiated by analyzinghDAGKβ expression by RT-PCR in cDNA from the cerebellum of normalindividuals and Alzheimer cerebellum. An initial analysis was carriedout using a set of hDAGKβ specific and SV-hDAGKβ specific primers andPCR conditions as detailed in the above section. The results (FIG. 4)indicate that expression of hDAGKβ is lost in Alzheimer cerebellum,while expression of SV-hDAGKβ is unchanged.

[0082] Screening for Compounds Which Exhibit hDAGKβ Modulating Activity

[0083] To identify modulators of hDAGK activity (especially hDAGKβ)activity like inhibitors and activators, respectively, a cellularhomogenate containing the hDAGK polypeptide (either from cellstransfected with the DAGK cDNA or from overproducing organisms) isincubated with substrates (like DAG and ATP) in the absence or thepresence of a chemical entity or crude natural extract that mightmodulate hDAGK activity (primary screening). The activity of the hDAGKpolypeptide or a purified preparation of hDAGK polypeptide in thereaction mixture can be quantified by measuring the ATP-dependentphosphorylation of the DAG substrate employing radio-labelled ATP assubstrate (20). The labelled product (phosphatidic acid) is extractedinto acidified organic solvents and quantitated by scintillationcounting. For more accurate determinations the phosphatidic acid productcan also be separated from the mixture by TLC methods and thecorresponding radioactive bands can be quantified by using aphosphoimager system. Fluorescence or chemiluminescence-tagged DAG canalso be used as the substrate for hDAGK. In this case, the PA productwill become labelled with the selected probe and can be separated fromthe substrate donor molecule by TLC and quantified via densitometricanalysis of fluorescent or chemoluminescent spots.

[0084] This application also relates to a method of identifying acompound or a composition that can activate or inhibit the activity ofthe promoter for the DNA of the present invention, which comprises (i)adding a test compound to a cell line whose hDAGK gene has beeninactivated by introducing a reporter gene, e.g., the beta-galactosidasefrom E. coli origin (lacZ); (ii) determining if transcription of thereporter gene occurs by measuring the level of activation of lacZ geneusing the chromogenic substrate X-gal using high-throughput calorimetricmeasurements. The compounds or compositions which are able to inhibit oractivate the promoter for the hDAGKβ DNA will alter the expression ofthe reporter gene. Compounds identified in this way are then tested invivo to assess their ability to modulate the level of the expression ofhDAGK, especially hDAGKβ, in mice CNS.

[0085] It is to be understood that modifications and/or alterations tothe aspects of the invention specifically disclosed within thisapplication, which based upon the disclosure herein would be readilyapparent to a person skilled in the art, are also considered to beincluded within the scope of the invention as outlined in the appendedclaims.

REFERENCES

[0086] 1. Sakane F., Kai, M., Wada, I., Imai, S., Kanoh, H. (1996). TheC-terminal part of diacylglycerol kinase α lacking zinc fingers servesas a catalytic domain. Biochem. J. 318, 583-590.

[0087] 2. Sakane, F. and Kanoh, H. (1997). Molecules in focus:diacylglycerol kinase. Int. J. Biochem. Cell Biol., 19, 1139-1143.

[0088] 3. Goto, K. and Kondo, H. (1993). Molecular cloning andexpression of a 90-kDa diacylglycerol kinase that predominantlylocalizes in neurons. Proc. Natl. Acad. Sci. 90, 7598-7602.

[0089] 4. Heimer, L., Zahm, D. S., Alheid, G. F. (1995). Basal ganglia.In: G. Paxinos “The rat nervous system” Academic Press, San Diego,pp.579-628

[0090] 5. Fisher, S. K., Heacock, A. M., Agranoff, B. W. (1992).Inositol lipids and signal transduction in the nervous system: anupdate. J. Neurochem. 58,18-38.

[0091] 6. Yuan, P X, Chen, G., Huang, L. D., Manji, H. K. (1998).Lithium stimulates gene expression through the AP-1 transcription factorpathway. Mol. Brain. Res. 58,225-230.

[0092] 7. Sambrook, J., Fritsch, E. F. and Maniatis, T. MolecularCloning: a Laboratory Manual. 2^(nd) Edition. CSH Laboratory Press.(1989)

[0093] 8. Boguski, M. S., Lowe, T. M. and Tolstohev, C. M. (1993) dbEST:database for ‘Expressed Sequence Tags’ Nature Genetics, 4, 332-333.

[0094] 9. Altschul, S. F., Warren, G., Webb, M., Myers, E. W., andLipman, D. J., (1990). Basic local alignment search tool. J. Mol. Biol.,215, 403-410.

[0095] 10. Wootton, J. C. and Federhen S., (1993). Statistics of localcomplexity in amino acid sequences and sequence databases. Comput. Chem.17,149-163

[0096] 11. Wootton, J. C. (1994). Non-globular domains in proteinsequences: aoutomated segmentation using complexity measures. Comput.Chem. 18, 269.

[0097] 12. Kamb, A., Wang, C., Thomas, A., DeHoff, B. S., Norris, F. H.,Richardson, K., Rine, J., Skolnick, M. H. and Rosteck Jr., P. R. (1995).Software trapping: a strategy for finding genes in large genomicregions. Comput. Biomed. Res. 28,140-153.

[0098] 13. Borodovsky, M. and Mc Ininch J. D., (1993). Parallel generecognition for both DNA strands. Computers and Chemistry 17, 123-133

[0099] 14. Uberbacher, E. C., and Mural, R. J., (1991). Locatingprotein-coding regions in Human DNA sequences by a multiplesensors-neural network approach. Proc. Nat!. Accad. Sci. USA, 88,11261-11265

[0100] 15. Schuler, G. D. (1998). Electronic PCR: bridging the gapbetween genome mapping and genome sequencing. Trends in Biotech. 16,456-459.

[0101] 16 Kawasaki E S. (1990). Amplification of RNA. In: PCR protocols,a guide to methods and applications. Eds Innis, Gelfand, Sninsky andWhite. Acad. Press.

[0102] 17. Trower M K., Burt D., Purvis I J., Dykes C W. & ChristodoulouC. (1995). Fluorescent dye-primer cycle sequencing using non-purifiedPCR products as templates; development of a protocol amenable tohigh-throughput DNA sequencing. Nucleic Acids Research, 23, 2348-2349

[0103] 18. Higgins, D. G. and Sharp, P. M. (1988). CLUSTAL: a packagefor performing multiple sequence alignments on a microcomputer. Gene 73,237-244.

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[0105] 20. Kanoh, H., Sakane, F., Yamada, K. (1992). Diacylglycerolkinase isozymes from brain and lymphoid tissues. Methods Enzymol. 209,162-172.

[0106]

1 7 1 3926 DNA Homo sapiens CDS (1)..(2415) 1 atg aca aac cag gaa aaatgg gcc cac ctc agc cct tcg gaa ttt tcc 48 Met Thr Asn Gln Glu Lys TrpAla His Leu Ser Pro Ser Glu Phe Ser 1 5 10 15 caa ctt cag aaa tat gctgag tat tct aca aag aaa tta aag gat gtt 96 Gln Leu Gln Lys Tyr Ala GluTyr Ser Thr Lys Lys Leu Lys Asp Val 20 25 30 ctt gaa gaa ttc cat ggt aatggt gtg ctt gca aag tat aat cct gaa 144 Leu Glu Glu Phe His Gly Asn GlyVal Leu Ala Lys Tyr Asn Pro Glu 35 40 45 ggg aaa caa gac att ctt aac caaaca ata gat ttt gaa ggt ttc aaa 192 Gly Lys Gln Asp Ile Leu Asn Gln ThrIle Asp Phe Glu Gly Phe Lys 50 55 60 cta ttc atg aag aca ttc ctg gaa gccgag ctt cct gat gat ttc act 240 Leu Phe Met Lys Thr Phe Leu Glu Ala GluLeu Pro Asp Asp Phe Thr 65 70 75 80 gca cac ctt ttc atg tca ttt agc aacaag ttt cct cat tct agt cca 288 Ala His Leu Phe Met Ser Phe Ser Asn LysPhe Pro His Ser Ser Pro 85 90 95 atg gta aaa agt aag cct gct ctc cta tcaggc ggt ctg aga atg aat 336 Met Val Lys Ser Lys Pro Ala Leu Leu Ser GlyGly Leu Arg Met Asn 100 105 110 aaa ggt gcc atc acc cct ccc cga act acttct cct gca aat acg tgt 384 Lys Gly Ala Ile Thr Pro Pro Arg Thr Thr SerPro Ala Asn Thr Cys 115 120 125 tcc cca gaa gta atc cat ctg aag gac attgtc tgt tac ctg tct ctg 432 Ser Pro Glu Val Ile His Leu Lys Asp Ile ValCys Tyr Leu Ser Leu 130 135 140 ctt gaa aga gga aga cct gag gat aag cttgag ttt atg ttt cgc ctt 480 Leu Glu Arg Gly Arg Pro Glu Asp Lys Leu GluPhe Met Phe Arg Leu 145 150 155 160 tat gac acg gat ggg aat ggc ttc ctggac agc tcg gag cta gaa aat 528 Tyr Asp Thr Asp Gly Asn Gly Phe Leu AspSer Ser Glu Leu Glu Asn 165 170 175 atc atc agt cag atg atg cat gtt gcagaa tac ctt gag tgg gat gtc 576 Ile Ile Ser Gln Met Met His Val Ala GluTyr Leu Glu Trp Asp Val 180 185 190 act gaa ctt aat cca atc ctc cat gaaatg atg gaa gaa att gac tat 624 Thr Glu Leu Asn Pro Ile Leu His Glu MetMet Glu Glu Ile Asp Tyr 195 200 205 gat cat gat gga acc gtg tct ctg gaggaa tgg att caa gga gga atg 672 Asp His Asp Gly Thr Val Ser Leu Glu GluTrp Ile Gln Gly Gly Met 210 215 220 aca acg att cca ctt ctt gtg ctc ctgggc tta gaa aat aac gtg aag 720 Thr Thr Ile Pro Leu Leu Val Leu Leu GlyLeu Glu Asn Asn Val Lys 225 230 235 240 gat gat gga cag cac gtg tgg cgactg aag cac ttt aac aaa cct gcc 768 Asp Asp Gly Gln His Val Trp Arg LeuLys His Phe Asn Lys Pro Ala 245 250 255 tat tgc aac ctt tgc ctg aac atgctg att ggc gtg ggg aag cag ggc 816 Tyr Cys Asn Leu Cys Leu Asn Met LeuIle Gly Val Gly Lys Gln Gly 260 265 270 ctc tgc tgt tcc ttc tgc aag tacaca gtc cat gag cgc tgt gtg gct 864 Leu Cys Cys Ser Phe Cys Lys Tyr ThrVal His Glu Arg Cys Val Ala 275 280 285 cga gca cct ccc tct tgc atc aagacc tat gtg aag tcc aaa agg aac 912 Arg Ala Pro Pro Ser Cys Ile Lys ThrTyr Val Lys Ser Lys Arg Asn 290 295 300 act gat gtc atg cac cat tac tgggtt gaa ggt aac tgc cca acc aag 960 Thr Asp Val Met His His Tyr Trp ValGlu Gly Asn Cys Pro Thr Lys 305 310 315 320 tgt gat aag tgc cac aaa actgtt aaa tgt tac cag ggc ctg aca gga 1008 Cys Asp Lys Cys His Lys Thr ValLys Cys Tyr Gln Gly Leu Thr Gly 325 330 335 ctg cat tgt gtt tgg tgt cagatc aca ctg cat aat aaa tgt gct tct 1056 Leu His Cys Val Trp Cys Gln IleThr Leu His Asn Lys Cys Ala Ser 340 345 350 cat cta aaa cct gaa tgt gactgt gga cct ttg aag gac cat att tta 1104 His Leu Lys Pro Glu Cys Asp CysGly Pro Leu Lys Asp His Ile Leu 355 360 365 cca ccc aca aca atc tgt ccagtg gta ctg cag act ctg ccc act tca 1152 Pro Pro Thr Thr Ile Cys Pro ValVal Leu Gln Thr Leu Pro Thr Ser 370 375 380 gga gtt tca gtt cct gag gaaaga caa tca aca gtg aaa aag gaa aag 1200 Gly Val Ser Val Pro Glu Glu ArgGln Ser Thr Val Lys Lys Glu Lys 385 390 395 400 agt ggt tcc cag cag ccaaac aaa gtg att gac aag aat aaa atg caa 1248 Ser Gly Ser Gln Gln Pro AsnLys Val Ile Asp Lys Asn Lys Met Gln 405 410 415 aga gcc aac tct gtt actgta gat gga caa ggc ctg cag gtc act cct 1296 Arg Ala Asn Ser Val Thr ValAsp Gly Gln Gly Leu Gln Val Thr Pro 420 425 430 gtg cct ggt act cac ccactt tta gtt ttt gtg aac ccc aaa agt ggt 1344 Val Pro Gly Thr His Pro LeuLeu Val Phe Val Asn Pro Lys Ser Gly 435 440 445 gga aaa caa gga gaa cgaatt tac aga aaa ttc cag tat cta tta aat 1392 Gly Lys Gln Gly Glu Arg IleTyr Arg Lys Phe Gln Tyr Leu Leu Asn 450 455 460 cct cgt cag gtt tac agtctt tct gga aat gga cca atg cca ggg tta 1440 Pro Arg Gln Val Tyr Ser LeuSer Gly Asn Gly Pro Met Pro Gly Leu 465 470 475 480 aac ttt ttc cgt gatgtt cct gac ttc aga gtg tta gcc tgt ggt gga 1488 Asn Phe Phe Arg Asp ValPro Asp Phe Arg Val Leu Ala Cys Gly Gly 485 490 495 gat gga acc gtg ggctgg gtt ttg gat tgc ata gaa aag gcc aat gta 1536 Asp Gly Thr Val Gly TrpVal Leu Asp Cys Ile Glu Lys Ala Asn Val 500 505 510 ggc aag cat cct ccagtt gcg att ctg cct ctt ggg act ggc aat gat 1584 Gly Lys His Pro Pro ValAla Ile Leu Pro Leu Gly Thr Gly Asn Asp 515 520 525 cta gca aga tgc ctgcga tgg gga gga ggt tac gaa ggt gag aat ctg 1632 Leu Ala Arg Cys Leu ArgTrp Gly Gly Gly Tyr Glu Gly Glu Asn Leu 530 535 540 atg aaa att cta aaagac att gaa aac agc aca gaa atc atg ttg gac 1680 Met Lys Ile Leu Lys AspIle Glu Asn Ser Thr Glu Ile Met Leu Asp 545 550 555 560 agg tgg aag tttgaa gtc ata cct aat gac aaa gat gag aaa gga gac 1728 Arg Trp Lys Phe GluVal Ile Pro Asn Asp Lys Asp Glu Lys Gly Asp 565 570 575 cca gtg cct tacagt atc atc aat aat tac ttt tcc att ggc gtg gat 1776 Pro Val Pro Tyr SerIle Ile Asn Asn Tyr Phe Ser Ile Gly Val Asp 580 585 590 gcc tcc att gcacac aga ttc cac atc atg aga gaa aaa cac cca gag 1824 Ala Ser Ile Ala HisArg Phe His Ile Met Arg Glu Lys His Pro Glu 595 600 605 aaa ttc aac agtaga atg aag aac aaa ttt tgg tat ttt gag ttt ggc 1872 Lys Phe Asn Ser ArgMet Lys Asn Lys Phe Trp Tyr Phe Glu Phe Gly 610 615 620 aca tct gaa actttc tca gcc acc tgc aag aag cta cat gaa tct gta 1920 Thr Ser Glu Thr PheSer Ala Thr Cys Lys Lys Leu His Glu Ser Val 625 630 635 640 gaa ata gaatgt gat gga gta cag ata gat tta ata aac atc tct ctg 1968 Glu Ile Glu CysAsp Gly Val Gln Ile Asp Leu Ile Asn Ile Ser Leu 645 650 655 gaa gga attgct att ttg aat ata cca agc atg cat gga gga tcc aat 2016 Glu Gly Ile AlaIle Leu Asn Ile Pro Ser Met His Gly Gly Ser Asn 660 665 670 ctt tgg ggagag tct aag aaa aga cga agc cat cga cga ata gag aaa 2064 Leu Trp Gly GluSer Lys Lys Arg Arg Ser His Arg Arg Ile Glu Lys 675 680 685 aaa ggg tctgac aaa agg acc acc gtc aca gat gcc aaa gag ttg aag 2112 Lys Gly Ser AspLys Arg Thr Thr Val Thr Asp Ala Lys Glu Leu Lys 690 695 700 ttt gca agtcaa gat ctc agt gac cag ctg ctg gag gtg gtc ggc ttg 2160 Phe Ala Ser GlnAsp Leu Ser Asp Gln Leu Leu Glu Val Val Gly Leu 705 710 715 720 gaa ggagcc atg gag atg ggg caa ata tac aca ggc ctg aaa agt gct 2208 Glu Gly AlaMet Glu Met Gly Gln Ile Tyr Thr Gly Leu Lys Ser Ala 725 730 735 ggc cggcgg ctg gct cag tgc tcc tgc gtg gtc atc agg acg agc aag 2256 Gly Arg ArgLeu Ala Gln Cys Ser Cys Val Val Ile Arg Thr Ser Lys 740 745 750 tct ctgcca atg caa att gat ggg gag cca tgg atg cag acc cca tgc 2304 Ser Leu ProMet Gln Ile Asp Gly Glu Pro Trp Met Gln Thr Pro Cys 755 760 765 aca ataaaa att aca cac aag aac caa gcc cca atg ctg atg ggc ccg 2352 Thr Ile LysIle Thr His Lys Asn Gln Ala Pro Met Leu Met Gly Pro 770 775 780 cct ccaaaa acc ggt tta ttc tgc tcc ctc gtc aaa agg aca aga aac 2400 Pro Pro LysThr Gly Leu Phe Cys Ser Leu Val Lys Arg Thr Arg Asn 785 790 795 800 cgaagc aag gaa taa tcctgtgttg tttcactctt agaaattgaa ttagcataat 2455 Arg SerLys Glu tgggccatgg aacacatatg ctggaaatct ttgaaccatt tcaagtctcctgctcatgca 2515 aaatcatgga agtggtttaa cagtttttgt tactaagcta atgtaaaattcagctattag 2575 aaaatttatt gtctcagttt ttataggcat ctttgcatga agaaagcagaagtttacctg 2635 aagtgatact gcatattttt ggtgcatgca ttcccataga tttttacatctcccacccaa 2695 ctcttcccca atttcctttt actaacctgt gagaaaaacc cgtgaaacatgaaaaaggaa 2755 ataccatggg aaacgtgatt ctcagtgtga ttccaattat tacgaagcactaatcagtaa 2815 cgctacaatg atcataattg cagattgcta tacgtttccc ttttagaatcagtgtatcag 2875 tgacctatga cttgaggaga aacttttaat tcgaagattt tattaaatagttgactacaa 2935 taccttgcta tatatacata gtttttcttc aacatcttaa ctcttctgagtggaaataaa 2995 aatatcaggc ataaggtttt ctcatgctga aaaatagaac gcggtttttattttgcttag 3055 ttttcttttt aattccagaa ataagtgaaa acatgttact tgacagtcaagtgtggtaat 3115 atggcaagcc ttgttccttt ctgcatgaga atctaggaga gaattcataaccacaccaat 3175 aacgaaatag aagttttaaa ctatgtgcct aatcaatgtg tttcccaccaaagattcaga 3235 aaacaatgct tgagagaaat gggttaatgc ataattaatt aagcattgtggagcaaattt 3295 agggttcctg tgattaattt tgtgatgact aaaatgctgg aaagcaagtgagttgcccat 3355 taattatgat taaaattctc acctttcaca gacagacaat aagccagacaacacaatcaa 3415 agctcaatag atgatttctt gcttttttca gtcatttata aatataggtgtaatttttca 3475 tggatcagtt aagtacactt gaaggaagta aatgattgta tcagtttatttctagtataa 3535 atgggtacct gtaataatac tgagctcttg gaagcgaatc atgcatgcaattagctccct 3595 cctcctcacc tactccactc ccatctttat gacatttcaa atgtttatttggaaacaaca 3655 gcctagatca ctgttgaagg tgttcatggc atagttggag tctctgactgtttaaagaaa 3715 tcacagaaca gtacttttct tttagtgttt cattaagcct atgatgtaaaatgaaatgct 3775 tctgagcagt cttgtaatat tgttcattca tattgacctg catctcatcattgcatgttt 3835 tatgttttca aacatgccat aaggaaaacg agtgcctgaa ctgcatgatttattagtttc 3895 tctccactct gcattaaagt gctaatgatt t 3926 2 804 PRT Homosapiens 2 Met Thr Asn Gln Glu Lys Trp Ala His Leu Ser Pro Ser Glu PheSer 1 5 10 15 Gln Leu Gln Lys Tyr Ala Glu Tyr Ser Thr Lys Lys Leu LysAsp Val 20 25 30 Leu Glu Glu Phe His Gly Asn Gly Val Leu Ala Lys Tyr AsnPro Glu 35 40 45 Gly Lys Gln Asp Ile Leu Asn Gln Thr Ile Asp Phe Glu GlyPhe Lys 50 55 60 Leu Phe Met Lys Thr Phe Leu Glu Ala Glu Leu Pro Asp AspPhe Thr 65 70 75 80 Ala His Leu Phe Met Ser Phe Ser Asn Lys Phe Pro HisSer Ser Pro 85 90 95 Met Val Lys Ser Lys Pro Ala Leu Leu Ser Gly Gly LeuArg Met Asn 100 105 110 Lys Gly Ala Ile Thr Pro Pro Arg Thr Thr Ser ProAla Asn Thr Cys 115 120 125 Ser Pro Glu Val Ile His Leu Lys Asp Ile ValCys Tyr Leu Ser Leu 130 135 140 Leu Glu Arg Gly Arg Pro Glu Asp Lys LeuGlu Phe Met Phe Arg Leu 145 150 155 160 Tyr Asp Thr Asp Gly Asn Gly PheLeu Asp Ser Ser Glu Leu Glu Asn 165 170 175 Ile Ile Ser Gln Met Met HisVal Ala Glu Tyr Leu Glu Trp Asp Val 180 185 190 Thr Glu Leu Asn Pro IleLeu His Glu Met Met Glu Glu Ile Asp Tyr 195 200 205 Asp His Asp Gly ThrVal Ser Leu Glu Glu Trp Ile Gln Gly Gly Met 210 215 220 Thr Thr Ile ProLeu Leu Val Leu Leu Gly Leu Glu Asn Asn Val Lys 225 230 235 240 Asp AspGly Gln His Val Trp Arg Leu Lys His Phe Asn Lys Pro Ala 245 250 255 TyrCys Asn Leu Cys Leu Asn Met Leu Ile Gly Val Gly Lys Gln Gly 260 265 270Leu Cys Cys Ser Phe Cys Lys Tyr Thr Val His Glu Arg Cys Val Ala 275 280285 Arg Ala Pro Pro Ser Cys Ile Lys Thr Tyr Val Lys Ser Lys Arg Asn 290295 300 Thr Asp Val Met His His Tyr Trp Val Glu Gly Asn Cys Pro Thr Lys305 310 315 320 Cys Asp Lys Cys His Lys Thr Val Lys Cys Tyr Gln Gly LeuThr Gly 325 330 335 Leu His Cys Val Trp Cys Gln Ile Thr Leu His Asn LysCys Ala Ser 340 345 350 His Leu Lys Pro Glu Cys Asp Cys Gly Pro Leu LysAsp His Ile Leu 355 360 365 Pro Pro Thr Thr Ile Cys Pro Val Val Leu GlnThr Leu Pro Thr Ser 370 375 380 Gly Val Ser Val Pro Glu Glu Arg Gln SerThr Val Lys Lys Glu Lys 385 390 395 400 Ser Gly Ser Gln Gln Pro Asn LysVal Ile Asp Lys Asn Lys Met Gln 405 410 415 Arg Ala Asn Ser Val Thr ValAsp Gly Gln Gly Leu Gln Val Thr Pro 420 425 430 Val Pro Gly Thr His ProLeu Leu Val Phe Val Asn Pro Lys Ser Gly 435 440 445 Gly Lys Gln Gly GluArg Ile Tyr Arg Lys Phe Gln Tyr Leu Leu Asn 450 455 460 Pro Arg Gln ValTyr Ser Leu Ser Gly Asn Gly Pro Met Pro Gly Leu 465 470 475 480 Asn PhePhe Arg Asp Val Pro Asp Phe Arg Val Leu Ala Cys Gly Gly 485 490 495 AspGly Thr Val Gly Trp Val Leu Asp Cys Ile Glu Lys Ala Asn Val 500 505 510Gly Lys His Pro Pro Val Ala Ile Leu Pro Leu Gly Thr Gly Asn Asp 515 520525 Leu Ala Arg Cys Leu Arg Trp Gly Gly Gly Tyr Glu Gly Glu Asn Leu 530535 540 Met Lys Ile Leu Lys Asp Ile Glu Asn Ser Thr Glu Ile Met Leu Asp545 550 555 560 Arg Trp Lys Phe Glu Val Ile Pro Asn Asp Lys Asp Glu LysGly Asp 565 570 575 Pro Val Pro Tyr Ser Ile Ile Asn Asn Tyr Phe Ser IleGly Val Asp 580 585 590 Ala Ser Ile Ala His Arg Phe His Ile Met Arg GluLys His Pro Glu 595 600 605 Lys Phe Asn Ser Arg Met Lys Asn Lys Phe TrpTyr Phe Glu Phe Gly 610 615 620 Thr Ser Glu Thr Phe Ser Ala Thr Cys LysLys Leu His Glu Ser Val 625 630 635 640 Glu Ile Glu Cys Asp Gly Val GlnIle Asp Leu Ile Asn Ile Ser Leu 645 650 655 Glu Gly Ile Ala Ile Leu AsnIle Pro Ser Met His Gly Gly Ser Asn 660 665 670 Leu Trp Gly Glu Ser LysLys Arg Arg Ser His Arg Arg Ile Glu Lys 675 680 685 Lys Gly Ser Asp LysArg Thr Thr Val Thr Asp Ala Lys Glu Leu Lys 690 695 700 Phe Ala Ser GlnAsp Leu Ser Asp Gln Leu Leu Glu Val Val Gly Leu 705 710 715 720 Glu GlyAla Met Glu Met Gly Gln Ile Tyr Thr Gly Leu Lys Ser Ala 725 730 735 GlyArg Arg Leu Ala Gln Cys Ser Cys Val Val Ile Arg Thr Ser Lys 740 745 750Ser Leu Pro Met Gln Ile Asp Gly Glu Pro Trp Met Gln Thr Pro Cys 755 760765 Thr Ile Lys Ile Thr His Lys Asn Gln Ala Pro Met Leu Met Gly Pro 770775 780 Pro Pro Lys Thr Gly Leu Phe Cys Ser Leu Val Lys Arg Thr Arg Asn785 790 795 800 Arg Ser Lys Glu 3 3926 DNA Homo sapiens 3 aaatcattagcactttaatg cagagtggag agaaactaat aaatcatgca gttcaggcac 60 tcgttttccttatggcatgt ttgaaaacat aaaacatgca atgatgagat gcaggtcaat 120 atgaatgaacaatattacaa gactgctcag aagcatttca ttttacatca taggcttaat 180 gaaacactaaaagaaaagta ctgttctgtg atttctttaa acagtcagag actccaacta 240 tgccatgaacaccttcaaca gtgatctagg ctgttgtttc caaataaaca tttgaaatgt 300 cataaagatgggagtggagt aggtgaggag gagggagcta attgcatgca tgattcgctt 360 ccaagagctcagtattatta caggtaccca tttatactag aaataaactg atacaatcat 420 ttacttccttcaagtgtact taactgatcc atgaaaaatt acacctatat ttataaatga 480 ctgaaaaaagcaagaaatca tctattgagc tttgattgtg ttgtctggct tattgtctgt 540 ctgtgaaaggtgagaatttt aatcataatt aatgggcaac tcacttgctt tccagcattt 600 tagtcatcacaaaattaatc acaggaaccc taaatttgct ccacaatgct taattaatta 660 tgcattaacccatttctctc aagcattgtt ttctgaatct ttggtgggaa acacattgat 720 taggcacatagtttaaaact tctatttcgt tattggtgtg gttatgaatt ctctcctaga 780 ttctcatgcagaaaggaaca aggcttgcca tattaccaca cttgactgtc aagtaacatg 840 ttttcacttatttctggaat taaaaagaaa actaagcaaa ataaaaaccg cgttctattt 900 ttcagcatgagaaaacctta tgcctgatat ttttatttcc actcagaaga gttaagatgt 960 tgaagaaaaactatgtatat atagcaaggt attgtagtca actatttaat aaaatcttcg 1020 aattaaaagtttctcctcaa gtcataggtc actgatacac tgattctaaa agggaaacgt 1080 atagcaatctgcaattatga tcattgtagc gttactgatt agtgcttcgt aataattgga 1140 atcacactgagaatcacgtt tcccatggta tttccttttt catgtttcac gggtttttct 1200 cacaggttagtaaaaggaaa ttggggaaga gttgggtggg agatgtaaaa atctatggga 1260 atgcatgcaccaaaaatatg cagtatcact tcaggtaaac ttctgctttc ttcatgcaaa 1320 gatgcctataaaaactgaga caataaattt tctaatagct gaattttaca ttagcttagt 1380 aacaaaaactgttaaaccac ttccatgatt ttgcatgagc aggagacttg aaatggttca 1440 aagatttccagcatatgtgt tccatggccc aattatgcta attcaatttc taagagtgaa 1500 acaacacaggattattcctt gcttcggttt cttgtccttt tgacgaggga gcagaataaa 1560 ccggtttttggaggcgggcc catcagcatt ggggcttggt tcttgtgtgt aatttttatt 1620 gtgcatggggtctgcatcca tggctcccca tcaatttgca ttggcagaga cttgctcgtc 1680 ctgatgaccacgcaggagca ctgagccagc cgccggccag cacttttcag gcctgtgtat 1740 atttgccccatctccatggc tccttccaag ccgaccacct ccagcagctg gtcactgaga 1800 tcttgacttgcaaacttcaa ctctttggca tctgtgacgg tggtcctttt gtcagaccct 1860 tttttctctattcgtcgatg gcttcgtctt ttcttagact ctccccaaag attggatcct 1920 ccatgcatgcttggtatatt caaaatagca attccttcca gagagatgtt tattaaatct 1980 atctgtactccatcacattc tatttctaca gattcatgta gcttcttgca ggtggctgag 2040 aaagtttcagatgtgccaaa ctcaaaatac caaaatttgt tcttcattct actgttgaat 2100 ttctctgggtgtttttctct catgatgtgg aatctgtgtg caatggaggc atccacgcca 2160 atggaaaagtaattattgat gatactgtaa ggcactgggt ctcctttctc atctttgtca 2220 ttaggtatgacttcaaactt ccacctgtcc aacatgattt ctgtgctgtt ttcaatgtct 2280 tttagaattttcatcagatt ctcaccttcg taacctcctc cccatcgcag gcatcttgct 2340 agatcattgccagtcccaag aggcagaatc gcaactggag gatgcttgcc tacattggcc 2400 ttttctatgcaatccaaaac ccagcccacg gttccatctc caccacaggc taacactctg 2460 aagtcaggaacatcacggaa aaagtttaac cctggcattg gtccatttcc agaaagactg 2520 taaacctgacgaggatttaa tagatactgg aattttctgt aaattcgttc tccttgtttt 2580 ccaccacttttggggttcac aaaaactaaa agtgggtgag taccaggcac aggagtgacc 2640 tgcaggccttgtccatctac agtaacagag ttggctcttt gcattttatt cttgtcaatc 2700 actttgtttggctgctggga accactcttt tcctttttca ctgttgattg tctttcctca 2760 ggaactgaaactcctgaagt gggcagagtc tgcagtacca ctggacagat tgttgtgggt 2820 ggtaaaatatggtccttcaa aggtccacag tcacattcag gttttagatg agaagcacat 2880 ttattatgcagtgtgatctg acaccaaaca caatgcagtc ctgtcaggcc ctggtaacat 2940 ttaacagttttgtggcactt atcacacttg gttgggcagt taccttcaac ccagtaatgg 3000 tgcatgacatcagtgttcct tttggacttc acataggtct tgatgcaaga gggaggtgct 3060 cgagccacacagcgctcatg gactgtgtac ttgcagaagg aacagcagag gccctgcttc 3120 cccacgccaatcagcatgtt caggcaaagg ttgcaatagg caggtttgtt aaagtgcttc 3180 agtcgccacacgtgctgtcc atcatccttc acgttatttt ctaagcccag gagcacaaga 3240 agtggaatcgttgtcattcc tccttgaatc cattcctcca gagacacggt tccatcatga 3300 tcatagtcaatttcttccat catttcatgg aggattggat taagttcagt gacatcccac 3360 tcaaggtattctgcaacatg catcatctga ctgatgatat tttctagctc cgagctgtcc 3420 aggaagccattcccatccgt gtcataaagg cgaaacataa actcaagctt atcctcaggt 3480 cttcctctttcaagcagaga caggtaacag acaatgtcct tcagatggat tacttctggg 3540 gaacacgtatttgcaggaga agtagttcgg ggaggggtga tggcaccttt attcattctc 3600 agaccgcctgataggagagc aggcttactt tttaccattg gactagaatg aggaaacttg 3660 ttgctaaatgacatgaaaag gtgtgcagtg aaatcatcag gaagctcggc ttccaggaat 3720 gtcttcatgaatagtttgaa accttcaaaa tctattgttt ggttaagaat gtcttgtttc 3780 ccttcaggattatactttgc aagcacacca ttaccatgga attcttcaag aacatccttt 3840 aatttctttgtagaatactc agcatatttc tgaagttggg aaaattccga agggctgagg 3900 tgggcccatttttcctggtt tgtcat 3926 4 3172 DNA Homo sapiens CDS (1)..(2322) 4 atg acaaac cag gaa aaa tgg gcc cac ctc agc cct tcg gaa ttt tcc 48 Met Thr AsnGln Glu Lys Trp Ala His Leu Ser Pro Ser Glu Phe Ser 1 5 10 15 caa cttcag aaa tat gct gag tat tct aca aag aaa tta aag gat gtt 96 Gln Leu GlnLys Tyr Ala Glu Tyr Ser Thr Lys Lys Leu Lys Asp Val 20 25 30 ctt gaa gaattc cat ggt aat ggt gtg ctt gca aag tat aat cct gaa 144 Leu Glu Glu PheHis Gly Asn Gly Val Leu Ala Lys Tyr Asn Pro Glu 35 40 45 ggg aaa caa gacatt ctt aac caa aca ata gat ttt gaa ggt ttc aaa 192 Gly Lys Gln Asp IleLeu Asn Gln Thr Ile Asp Phe Glu Gly Phe Lys 50 55 60 cta ttc atg aag acattc ctg gaa gcc gag ctt cct gat gat ttc act 240 Leu Phe Met Lys Thr PheLeu Glu Ala Glu Leu Pro Asp Asp Phe Thr 65 70 75 80 gca cac ctt ttc atgtca ttt agc aac aag ttt cct cat tct agt cca 288 Ala His Leu Phe Met SerPhe Ser Asn Lys Phe Pro His Ser Ser Pro 85 90 95 atg gta aaa agt aag cctgct ctc cta tca ggc ggt ctg aga atg aat 336 Met Val Lys Ser Lys Pro AlaLeu Leu Ser Gly Gly Leu Arg Met Asn 100 105 110 aaa ggt gcc atc acc cctccc cga act act tct cct gca aat acg tgt 384 Lys Gly Ala Ile Thr Pro ProArg Thr Thr Ser Pro Ala Asn Thr Cys 115 120 125 tcc cca gaa gta atc catctg aag gac att gtc tgt tac ctg tct ctg 432 Ser Pro Glu Val Ile His LeuLys Asp Ile Val Cys Tyr Leu Ser Leu 130 135 140 ctt gaa aga gga aga cctgag gat aag ctt gag ttt atg ttt cgc ctt 480 Leu Glu Arg Gly Arg Pro GluAsp Lys Leu Glu Phe Met Phe Arg Leu 145 150 155 160 tat gac acg gat gggaat ggc ttc ctg gac agc tcg gag cta gaa aat 528 Tyr Asp Thr Asp Gly AsnGly Phe Leu Asp Ser Ser Glu Leu Glu Asn 165 170 175 atc atc agt cag atgatg cat gtt gca gaa tac ctt gag tgg gat gtc 576 Ile Ile Ser Gln Met MetHis Val Ala Glu Tyr Leu Glu Trp Asp Val 180 185 190 act gaa ctt aat ccaatc ctc cat gaa atg atg gaa gaa att gac tat 624 Thr Glu Leu Asn Pro IleLeu His Glu Met Met Glu Glu Ile Asp Tyr 195 200 205 gat cat gat gga accgtg tct ctg gag gaa tgg att caa gga gga atg 672 Asp His Asp Gly Thr ValSer Leu Glu Glu Trp Ile Gln Gly Gly Met 210 215 220 aca acg att cca cttctt gtg ctc ctg ggc tta gaa aat aac gtg aag 720 Thr Thr Ile Pro Leu LeuVal Leu Leu Gly Leu Glu Asn Asn Val Lys 225 230 235 240 gat gat gga cagcac gtg tgg cga ctg aag cac ttt aac aaa cct gcc 768 Asp Asp Gly Gln HisVal Trp Arg Leu Lys His Phe Asn Lys Pro Ala 245 250 255 tat tgc aac ctttgc ctg aac atg ctg att ggc gtg ggg aag cag ggc 816 Tyr Cys Asn Leu CysLeu Asn Met Leu Ile Gly Val Gly Lys Gln Gly 260 265 270 ctc tgc tgt tccttc tgc aag tac aca gtc cat gag cgc tgt gtg gct 864 Leu Cys Cys Ser PheCys Lys Tyr Thr Val His Glu Arg Cys Val Ala 275 280 285 cga gca cct ccctct tgc atc aag acc tat gtg aag tcc aaa agg aac 912 Arg Ala Pro Pro SerCys Ile Lys Thr Tyr Val Lys Ser Lys Arg Asn 290 295 300 act gat gtc atgcac cat tac tgg gtt gaa ggt aac tgc cca acc aag 960 Thr Asp Val Met HisHis Tyr Trp Val Glu Gly Asn Cys Pro Thr Lys 305 310 315 320 tgt gat aagtgc cac aaa act gtt aaa tgt tac cag ggc ctg aca gga 1008 Cys Asp Lys CysHis Lys Thr Val Lys Cys Tyr Gln Gly Leu Thr Gly 325 330 335 ctg cat tgtgtt tgg tgt cag atc aca ctg cat aat aaa tgt gct tct 1056 Leu His Cys ValTrp Cys Gln Ile Thr Leu His Asn Lys Cys Ala Ser 340 345 350 cat cta aaacct gaa tgt gac tgt gga cct ttg aag gac cat att tta 1104 His Leu Lys ProGlu Cys Asp Cys Gly Pro Leu Lys Asp His Ile Leu 355 360 365 cca ccc acaaca atc tgt cca gtg gta ctg cag act ctg ccc act tca 1152 Pro Pro Thr ThrIle Cys Pro Val Val Leu Gln Thr Leu Pro Thr Ser 370 375 380 gga gtt tcagtt cct gag gaa aga caa tca aca gtg aaa aag gaa aag 1200 Gly Val Ser ValPro Glu Glu Arg Gln Ser Thr Val Lys Lys Glu Lys 385 390 395 400 agt ggttcc cag cag cca aac aaa gtg att gac aag aat aaa atg caa 1248 Ser Gly SerGln Gln Pro Asn Lys Val Ile Asp Lys Asn Lys Met Gln 405 410 415 aga gccaac tct gtt act gta gat gga caa ggc ctg cag gtc act cct 1296 Arg Ala AsnSer Val Thr Val Asp Gly Gln Gly Leu Gln Val Thr Pro 420 425 430 gtg cctggt act cac cca ctt tta gtt ttt gtg aac ccc aaa agt ggt 1344 Val Pro GlyThr His Pro Leu Leu Val Phe Val Asn Pro Lys Ser Gly 435 440 445 gga aaacaa gga gaa cga att tac aga aaa ttc cag tat cta tta aat 1392 Gly Lys GlnGly Glu Arg Ile Tyr Arg Lys Phe Gln Tyr Leu Leu Asn 450 455 460 cct cgtcag gtt tac agt ctt tct gga aat gga cca atg cca ggg tta 1440 Pro Arg GlnVal Tyr Ser Leu Ser Gly Asn Gly Pro Met Pro Gly Leu 465 470 475 480 aacttt ttc cgt gat gtt cct gac ttc aga gtg tta gcc tgt ggt gga 1488 Asn PhePhe Arg Asp Val Pro Asp Phe Arg Val Leu Ala Cys Gly Gly 485 490 495 gatgga acc gtg ggc tgg gtt ttg gat tgc ata gaa aag gcc aat gta 1536 Asp GlyThr Val Gly Trp Val Leu Asp Cys Ile Glu Lys Ala Asn Val 500 505 510 ggcaag cat cct cca gtt gcg att ctg cct ctt ggg act ggc aat gat 1584 Gly LysHis Pro Pro Val Ala Ile Leu Pro Leu Gly Thr Gly Asn Asp 515 520 525 ctagca aga tgc ctg cga tgg gga gga ggt tac gaa ggt gag aat ctg 1632 Leu AlaArg Cys Leu Arg Trp Gly Gly Gly Tyr Glu Gly Glu Asn Leu 530 535 540 atgaaa att cta aaa gac att gaa aac agc aca gaa atc atg ttg gac 1680 Met LysIle Leu Lys Asp Ile Glu Asn Ser Thr Glu Ile Met Leu Asp 545 550 555 560agg tgg aag ttt gaa gtc ata cct aat gac aaa gat gag aaa gga gac 1728 ArgTrp Lys Phe Glu Val Ile Pro Asn Asp Lys Asp Glu Lys Gly Asp 565 570 575cca gtg cct tac agt atc atc aat aat tac ttt tcc att ggc gtg gat 1776 ProVal Pro Tyr Ser Ile Ile Asn Asn Tyr Phe Ser Ile Gly Val Asp 580 585 590gcc tcc att gca cac aga ttc cac atc atg aga gaa aaa cac cca gag 1824 AlaSer Ile Ala His Arg Phe His Ile Met Arg Glu Lys His Pro Glu 595 600 605aaa ttc aac agt aga atg aag aac aaa ttt tgg tat ttt gag ttt ggc 1872 LysPhe Asn Ser Arg Met Lys Asn Lys Phe Trp Tyr Phe Glu Phe Gly 610 615 620aca tct gaa act ttc tca gcc acc tgc aag aag cta cat gaa tct gta 1920 ThrSer Glu Thr Phe Ser Ala Thr Cys Lys Lys Leu His Glu Ser Val 625 630 635640 gaa ata gaa tgt gat gga gta cag ata gat tta ata aac atc tct ctg 1968Glu Ile Glu Cys Asp Gly Val Gln Ile Asp Leu Ile Asn Ile Ser Leu 645 650655 gaa gga att gct att ttg aat ata cca agc atg cat gga gga tcc aat 2016Glu Gly Ile Ala Ile Leu Asn Ile Pro Ser Met His Gly Gly Ser Asn 660 665670 ctt tgg gga gag tct aag aaa aga cga agc cat cga cga ata gag aaa 2064Leu Trp Gly Glu Ser Lys Lys Arg Arg Ser His Arg Arg Ile Glu Lys 675 680685 aaa ggg tct gac aaa agg acc acc gtc aca gat gcc aaa gag ttg aag 2112Lys Gly Ser Asp Lys Arg Thr Thr Val Thr Asp Ala Lys Glu Leu Lys 690 695700 ttt gca agt caa gat ctc agt gac cag ctg ctg gag gtg gtc ggc ttg 2160Phe Ala Ser Gln Asp Leu Ser Asp Gln Leu Leu Glu Val Val Gly Leu 705 710715 720 gaa gga gcc atg gag atg ggg caa ata tac aca ggc ctg aaa agt gct2208 Glu Gly Ala Met Glu Met Gly Gln Ile Tyr Thr Gly Leu Lys Ser Ala 725730 735 ggc cgg cgg ctg gct cag tgc tcc tgc gtg gtc atc agg acg agc aag2256 Gly Arg Arg Leu Ala Gln Cys Ser Cys Val Val Ile Arg Thr Ser Lys 740745 750 tct ctg cca atg caa att gat ggg gag cca tgg atg cag acc cca tgc2304 Ser Leu Pro Met Gln Ile Asp Gly Glu Pro Trp Met Gln Thr Pro Cys 755760 765 aca gtg agt aca gag tag ttgatatgct atgtcaatct cagttttgct 2352Thr Val Ser Thr Glu 770 ttcctctttg actaaataac cacaataact gatttttttctttatttctt ttcaacctat 2412 cagcaaatag tctttttgtt gttgttgtta tgtgtgtgtcagagccacta catttaggct 2472 gtagacatta tatacccttg gcaatgattt agctcttgaatgtttgtgct agcctaagta 2532 taaatagatc ttttaaatag atcaattata aaccatagatcaattataaa ctatggagct 2592 aaacaaaata ttaataaaag tttatctgaa acttttttgtttatttcaga gcacattatt 2652 agaatattat ttgcgagaaa tgcagaccta agcttatatgtgaacttatt tctcagcttt 2712 tctatgcctc catttgggga tttgagggct ttcttctccataagaaaaaa atttctctcc 2772 agtttctacc ataattaatt gtgttttcca gaatgaggtattatttaagg cagacactgc 2832 ccctctcaaa aaaaatcagt tttcatttgc atagtgaatattttattgca tttcaaaaac 2892 atgctaggaa ctgcttttgg cactgggagt agacacatgaacaagaccaa cagtgtaatt 2952 tccttcaagt tacttacatt cctataatag aggaccgaataaataaacaa ctacatgata 3012 aatataactt cagactgtga gagttattaa aaaataaggtgaaatgatga taagaagctg 3072 gattaggtgt ggagaataaa tactacttga gataagggagacctctttga aaggacatag 3132 ccaaaagctt agtataaaat taaaaaaaat aaaaaaaaaa3172 5 773 PRT Homo sapiens 5 Met Thr Asn Gln Glu Lys Trp Ala His LeuSer Pro Ser Glu Phe Ser 1 5 10 15 Gln Leu Gln Lys Tyr Ala Glu Tyr SerThr Lys Lys Leu Lys Asp Val 20 25 30 Leu Glu Glu Phe His Gly Asn Gly ValLeu Ala Lys Tyr Asn Pro Glu 35 40 45 Gly Lys Gln Asp Ile Leu Asn Gln ThrIle Asp Phe Glu Gly Phe Lys 50 55 60 Leu Phe Met Lys Thr Phe Leu Glu AlaGlu Leu Pro Asp Asp Phe Thr 65 70 75 80 Ala His Leu Phe Met Ser Phe SerAsn Lys Phe Pro His Ser Ser Pro 85 90 95 Met Val Lys Ser Lys Pro Ala LeuLeu Ser Gly Gly Leu Arg Met Asn 100 105 110 Lys Gly Ala Ile Thr Pro ProArg Thr Thr Ser Pro Ala Asn Thr Cys 115 120 125 Ser Pro Glu Val Ile HisLeu Lys Asp Ile Val Cys Tyr Leu Ser Leu 130 135 140 Leu Glu Arg Gly ArgPro Glu Asp Lys Leu Glu Phe Met Phe Arg Leu 145 150 155 160 Tyr Asp ThrAsp Gly Asn Gly Phe Leu Asp Ser Ser Glu Leu Glu Asn 165 170 175 Ile IleSer Gln Met Met His Val Ala Glu Tyr Leu Glu Trp Asp Val 180 185 190 ThrGlu Leu Asn Pro Ile Leu His Glu Met Met Glu Glu Ile Asp Tyr 195 200 205Asp His Asp Gly Thr Val Ser Leu Glu Glu Trp Ile Gln Gly Gly Met 210 215220 Thr Thr Ile Pro Leu Leu Val Leu Leu Gly Leu Glu Asn Asn Val Lys 225230 235 240 Asp Asp Gly Gln His Val Trp Arg Leu Lys His Phe Asn Lys ProAla 245 250 255 Tyr Cys Asn Leu Cys Leu Asn Met Leu Ile Gly Val Gly LysGln Gly 260 265 270 Leu Cys Cys Ser Phe Cys Lys Tyr Thr Val His Glu ArgCys Val Ala 275 280 285 Arg Ala Pro Pro Ser Cys Ile Lys Thr Tyr Val LysSer Lys Arg Asn 290 295 300 Thr Asp Val Met His His Tyr Trp Val Glu GlyAsn Cys Pro Thr Lys 305 310 315 320 Cys Asp Lys Cys His Lys Thr Val LysCys Tyr Gln Gly Leu Thr Gly 325 330 335 Leu His Cys Val Trp Cys Gln IleThr Leu His Asn Lys Cys Ala Ser 340 345 350 His Leu Lys Pro Glu Cys AspCys Gly Pro Leu Lys Asp His Ile Leu 355 360 365 Pro Pro Thr Thr Ile CysPro Val Val Leu Gln Thr Leu Pro Thr Ser 370 375 380 Gly Val Ser Val ProGlu Glu Arg Gln Ser Thr Val Lys Lys Glu Lys 385 390 395 400 Ser Gly SerGln Gln Pro Asn Lys Val Ile Asp Lys Asn Lys Met Gln 405 410 415 Arg AlaAsn Ser Val Thr Val Asp Gly Gln Gly Leu Gln Val Thr Pro 420 425 430 ValPro Gly Thr His Pro Leu Leu Val Phe Val Asn Pro Lys Ser Gly 435 440 445Gly Lys Gln Gly Glu Arg Ile Tyr Arg Lys Phe Gln Tyr Leu Leu Asn 450 455460 Pro Arg Gln Val Tyr Ser Leu Ser Gly Asn Gly Pro Met Pro Gly Leu 465470 475 480 Asn Phe Phe Arg Asp Val Pro Asp Phe Arg Val Leu Ala Cys GlyGly 485 490 495 Asp Gly Thr Val Gly Trp Val Leu Asp Cys Ile Glu Lys AlaAsn Val 500 505 510 Gly Lys His Pro Pro Val Ala Ile Leu Pro Leu Gly ThrGly Asn Asp 515 520 525 Leu Ala Arg Cys Leu Arg Trp Gly Gly Gly Tyr GluGly Glu Asn Leu 530 535 540 Met Lys Ile Leu Lys Asp Ile Glu Asn Ser ThrGlu Ile Met Leu Asp 545 550 555 560 Arg Trp Lys Phe Glu Val Ile Pro AsnAsp Lys Asp Glu Lys Gly Asp 565 570 575 Pro Val Pro Tyr Ser Ile Ile AsnAsn Tyr Phe Ser Ile Gly Val Asp 580 585 590 Ala Ser Ile Ala His Arg PheHis Ile Met Arg Glu Lys His Pro Glu 595 600 605 Lys Phe Asn Ser Arg MetLys Asn Lys Phe Trp Tyr Phe Glu Phe Gly 610 615 620 Thr Ser Glu Thr PheSer Ala Thr Cys Lys Lys Leu His Glu Ser Val 625 630 635 640 Glu Ile GluCys Asp Gly Val Gln Ile Asp Leu Ile Asn Ile Ser Leu 645 650 655 Glu GlyIle Ala Ile Leu Asn Ile Pro Ser Met His Gly Gly Ser Asn 660 665 670 LeuTrp Gly Glu Ser Lys Lys Arg Arg Ser His Arg Arg Ile Glu Lys 675 680 685Lys Gly Ser Asp Lys Arg Thr Thr Val Thr Asp Ala Lys Glu Leu Lys 690 695700 Phe Ala Ser Gln Asp Leu Ser Asp Gln Leu Leu Glu Val Val Gly Leu 705710 715 720 Glu Gly Ala Met Glu Met Gly Gln Ile Tyr Thr Gly Leu Lys SerAla 725 730 735 Gly Arg Arg Leu Ala Gln Cys Ser Cys Val Val Ile Arg ThrSer Lys 740 745 750 Ser Leu Pro Met Gln Ile Asp Gly Glu Pro Trp Met GlnThr Pro Cys 755 760 765 Thr Val Ser Thr Glu 770 6 3172 DNA Homo sapiens6 tttttttttt atttttttta attttatact aagcttttgg ctatgtcctt tcaaagaggt 60ctcccttatc tcaagtagta tttattctcc acacctaatc cagcttctta tcatcatttc 120accttatttt ttaataactc tcacagtctg aagttatatt tatcatgtag ttgtttattt 180attcggtcct ctattatagg aatgtaagta acttgaagga aattacactg ttggtcttgt 240tcatgtgtct actcccagtg ccaaaagcag ttcctagcat gtttttgaaa tgcaataaaa 300tattcactat gcaaatgaaa actgattttt tttgagaggg gcagtgtctg ccttaaataa 360tacctcattc tggaaaacac aattaattat ggtagaaact ggagagaaat ttttttctta 420tggagaagaa agccctcaaa tccccaaatg gaggcataga aaagctgaga aataagttca 480catataagct taggtctgca tttctcgcaa ataatattct aataatgtgc tctgaaataa 540acaaaaaagt ttcagataaa cttttattaa tattttgttt agctccatag tttataattg 600atctatggtt tataattgat ctatttaaaa gatctattta tacttaggct agcacaaaca 660ttcaagagct aaatcattgc caagggtata taatgtctac agcctaaatg tagtggctct 720gacacacaca taacaacaac aacaaaaaga ctatttgctg ataggttgaa aagaaataaa 780gaaaaaaatc agttattgtg gttatttagt caaagaggaa agcaaaactg agattgacat 840agcatatcaa ctactctgta ctcactgtgc atggggtctg catccatggc tccccatcaa 900tttgcattgg cagagacttg ctcgtcctga tgaccacgca ggagcactga gccagccgcc 960ggccagcact tttcaggcct gtgtatattt gccccatctc catggctcct tccaagccga 1020ccacctccag cagctggtca ctgagatctt gacttgcaaa cttcaactct ttggcatctg 1080tgacggtggt ccttttgtca gacccttttt tctctattcg tcgatggctt cgtcttttct 1140tagactctcc ccaaagattg gatcctccat gcatgcttgg tatattcaaa atagcaattc 1200cttccagaga gatgtttatt aaatctatct gtactccatc acattctatt tctacagatt 1260catgtagctt cttgcaggtg gctgagaaag tttcagatgt gccaaactca aaataccaaa 1320atttgttctt cattctactg ttgaatttct ctgggtgttt ttctctcatg atgtggaatc 1380tgtgtgcaat ggaggcatcc acgccaatgg aaaagtaatt attgatgata ctgtaaggca 1440ctgggtctcc tttctcatct ttgtcattag gtatgacttc aaacttccac ctgtccaaca 1500tgatttctgt gctgttttca atgtctttta gaattttcat cagattctca ccttcgtaac 1560ctcctcccca tcgcaggcat cttgctagat cattgccagt cccaagaggc agaatcgcaa 1620ctggaggatg cttgcctaca ttggcctttt ctatgcaatc caaaacccag cccacggttc 1680catctccacc acaggctaac actctgaagt caggaacatc acggaaaaag tttaaccctg 1740gcattggtcc atttccagaa agactgtaaa cctgacgagg atttaataga tactggaatt 1800ttctgtaaat tcgttctcct tgttttccac cacttttggg gttcacaaaa actaaaagtg 1860ggtgagtacc aggcacagga gtgacctgca ggccttgtcc atctacagta acagagttgg 1920ctctttgcat tttattcttg tcaatcactt tgtttggctg ctgggaacca ctcttttcct 1980ttttcactgt tgattgtctt tcctcaggaa ctgaaactcc tgaagtgggc agagtctgca 2040gtaccactgg acagattgtt gtgggtggta aaatatggtc cttcaaaggt ccacagtcac 2100attcaggttt tagatgagaa gcacatttat tatgcagtgt gatctgacac caaacacaat 2160gcagtcctgt caggccctgg taacatttaa cagttttgtg gcacttatca cacttggttg 2220ggcagttacc ttcaacccag taatggtgca tgacatcagt gttccttttg gacttcacat 2280aggtcttgat gcaagaggga ggtgctcgag ccacacagcg ctcatggact gtgtacttgc 2340agaaggaaca gcagaggccc tgcttcccca cgccaatcag catgttcagg caaaggttgc 2400aataggcagg tttgttaaag tgcttcagtc gccacacgtg ctgtccatca tccttcacgt 2460tattttctaa gcccaggagc acaagaagtg gaatcgttgt cattcctcct tgaatccatt 2520cctccagaga cacggttcca tcatgatcat agtcaatttc ttccatcatt tcatggagga 2580ttggattaag ttcagtgaca tcccactcaa ggtattctgc aacatgcatc atctgactga 2640tgatattttc tagctccgag ctgtccagga agccattccc atccgtgtca taaaggcgaa 2700acataaactc aagcttatcc tcaggtcttc ctctttcaag cagagacagg taacagacaa 2760tgtccttcag atggattact tctggggaac acgtatttgc aggagaagta gttcggggag 2820gggtgatggc acctttattc attctcagac cgcctgatag gagagcaggc ttacttttta 2880ccattggact agaatgagga aacttgttgc taaatgacat gaaaaggtgt gcagtgaaat 2940catcaggaag ctcggcttcc aggaatgtct tcatgaatag tttgaaacct tcaaaatcta 3000ttgtttggtt aagaatgtct tgtttccctt caggattata ctttgcaagc acaccattac 3060catggaattc ttcaagaaca tcctttaatt tctttgtaga atactcagca tatttctgaa 3120gttgggaaaa ttccgaaggg ctgaggtggg cccatttttc ctggtttgtc at 3172 7 801 PRTRattus sp. 7 Met Thr Asn Gln Glu Lys Trp Ala His Leu Ser Pro Ser Glu PheSer 1 5 10 15 Gln Leu Gln Lys Tyr Ala Glu Tyr Ser Thr Lys Lys Leu LysAsp Val 20 25 30 Leu Glu Glu Phe His Gly Asn Gly Val Leu Ala Lys Tyr AsnPro Glu 35 40 45 Gly Lys Gln Asp Ile Leu Asn Gln Thr Ile Asp Phe Glu GlyPhe Lys 50 55 60 Leu Phe Met Lys Thr Phe Leu Glu Ala Glu Leu Pro Asp AspPhe Thr 65 70 75 80 Ala His Leu Phe Met Ser Phe Ser Asn Lys Phe Pro HisSer Ser Pro 85 90 95 Asn Val Lys Ser Lys Pro Ala Leu Leu Ser Gly Gly LeuArg Met Asn 100 105 110 Lys Gly Ala Ile Thr Pro Pro Arg Ser Ser Pro AlaAsn Thr Cys Phe 115 120 125 Pro Glu Val Ile His Leu Lys Asp Ile Val CysTyr Leu Ser Leu Leu 130 135 140 Glu Arg Gly Arg Pro Glu Asp Lys Leu GluPhe Met Phe Arg Leu Tyr 145 150 155 160 Asp Thr Asp Gly Asn Gly Phe LeuAsp Ser Ser Glu Leu Glu Asn Ile 165 170 175 Ile Gly Gln Met Met His ValAla Glu Tyr Leu Glu Trp Asp Val Thr 180 185 190 Glu Leu Asn Pro Ile LeuHis Glu Met Met Glu Glu Ile Asp Tyr Asp 195 200 205 Arg Asp Gly Thr ValSer Leu Glu Glu Trp Ile Gln Gly Gly Met Thr 210 215 220 Thr Ile Pro LeuLeu Val Leu Leu Gly Leu Glu Asn Asn Val Lys Asp 225 230 235 240 Asp GlyGln His Val Trp Arg Leu Lys His Phe Asn Lys Pro Ala Tyr 245 250 255 CysAsn Leu Cys Leu Asn Met Leu Ile Gly Val Gly Lys Gln Gly Leu 260 265 270Cys Cys Ser Phe Cys Lys Tyr Thr Val His Glu Arg Cys Ala Arg Ala 275 280285 Pro Pro Ser Cys Ile Lys Thr Tyr Val Lys Ser Lys Lys Asn Thr Asp 290295 300 Val Met His His Tyr Trp Val Glu Gly Asn Cys Pro Thr Lys Cys Asp305 310 315 320 Lys Cys His Lys Thr Val Lys Cys Tyr Gln Gly Leu Thr GlyLeu His 325 330 335 Cys Val Trp Cys Gln Thr Thr Leu His Asn Lys Cys AlaSer His Leu 340 345 350 Lys Pro Glu Cys Asp Cys Gly Pro Leu Lys Asp HisIle Leu Pro Pro 355 360 365 Thr Thr Ile Cys Pro Val Val Leu Thr Met ProThr Ala Gly Thr Ser 370 375 380 Val Pro Glu Glu Arg Gln Ser Thr Ala LysLys Glu Lys Gly Ser Ser 385 390 395 400 Gln Gln Pro Asn Lys Val Thr AspLys Asn Lys Met Gln Arg Ala Asn 405 410 415 Ser Val Thr Met Asp Gly GlnGly Leu Gln Ile Thr Pro Ile Pro Gly 420 425 430 Thr His Pro Leu Leu ValPhe Val Asn Pro Lys Ser Gly Gly Lys Gln 435 440 445 Gly Glu Arg Ile TyrArg Lys Phe Gln Tyr Leu Leu Asn Pro Arg Gln 450 455 460 Val Tyr Ser LeuSer Gly Asn Gly Pro Met Pro Gly Leu His Phe Phe 465 470 475 480 Arg AspVal Pro Asp Phe Arg Val Leu Ala Cys Gly Gly Asp Gly Thr 485 490 495 ValGly Trp Ile Leu Asp Cys Ile Glu Lys Ala Asn Val Val Lys His 500 505 510Pro Pro Val Ala Ile Leu Pro Leu Gly Thr Gly Asn Asp Leu Ala Arg 515 520525 Cys Leu Arg Trp Gly Gly Gly Tyr Glu Gly Glu Asn Leu Met Lys Ile 530535 540 Leu Lys Asp Ile Glu Ser Ser Thr Glu Ile Met Leu Asp Arg Trp Lys545 550 555 560 Phe Glu Val Thr Pro Asn Asp Lys Asp Glu Lys Gly Asp ProVal Pro 565 570 575 Tyr Ser Ile Ile Asn Asn Tyr Phe Ser Ile Gly Val AspAla Ser Ile 580 585 590 Ala His Arg Phe His Ile Met Arg Glu Lys His ProGlu Lys Phe Asn 595 600 605 Ser Arg Met Lys Asn Lys Phe Trp Tyr Phe GluPhe Gly Thr Ser Glu 610 615 620 Thr Phe Ser Ala Thr Cys Lys Lys Leu HisGlu Ser Val Glu Ile Glu 625 630 635 640 Cys Asp Gly Val Gln Ile Asp LeuIle Asn Ile Ser Leu Gln Gly Ile 645 650 655 Ala Ile Leu Asn Ile Pro SerMet His Gly Gly Ser Asn Leu Trp Gly 660 665 670 Glu Ser Lys Lys Lys ArgSer His Arg Arg Ile Glu Lys Lys Gly Ser 675 680 685 Asp Lys Arg Pro ThrLeu Thr Asp Ala Lys Glu Leu Lys Phe Ala Ser 690 695 700 Gln Asp Leu SerAsp Gln Leu Leu Glu Val Val Gly Leu Glu Gly Ala 705 710 715 720 Met GluMet Gly Gln Ile Tyr Thr Gly Leu Lys Ser Ala Gly Arg Arg 725 730 735 LeuAla Gln Cys Ser Ser Val Val Ile Arg Thr Ser Lys Ser Leu Pro 740 745 750Met Gln Ile Asp Gly Glu Pro Trp Met Gln Thr Pro Cys Thr Ile Lys 755 760765 Ile Thr His Lys Asn Gln Ala Pro Met Leu Met Gly Pro Pro Pro Lys 770775 780 Thr Gly Leu Phe Cys Ser Leu Ile Lys Arg Thr Arg Asn Arg Ser Lys785 790 795 800 Glu

1. An isolated human diacylglycerol kinase β (hDAGKβ) protein or avariant thereof.
 2. hDAGKβ protein according to claim 1 having an aminoacid sequence as set out in Seq ID No 1 or in Seq ID No
 4. 3. hDAGKβprotein according to claim 1 having an amino acid sequence as set out inSeq ID No
 4. 4. A nucleotide sequence encoding a hDAGKβ protein or avariant thereof, or a nucleotide sequence which is complementarythereto.
 5. The nucleotide sequence according to claim 4 having asequence as set out in Seq ID No 3 or in Seq ID No
 6. 6. The nucleotidesequence according to claim 4 having a sequence as set out in Seq ID No6.
 7. The nucleotide sequence according to in any of claims 4 to 6 orwhich is a cDNA sequence.
 8. An expression vector comprising anucleotide sequence according to any one of claims 4 to 7, which iscapable of expressing an hDAGKβ protein.
 9. A stable cell linecomprising an expression vector according to claim
 8. 10. The cell lineaccording to claim 9 that is a modified HEK293, COS or HeLa cell line.11. An antibody specific for a protein as claimed in any claims 1 to 3.12. A method for identification of a compound that exhibits DAGKmodulating activity, comprising contacting a DAGK protein with a testcompound and detecting modulation of enzyme activity or detecting enzymeinactivity.
 13. The method according to claim 12 wherein the DAGKprotein is hDAGKβ.
 14. A compound that modulates hDAGK activity,identifiable by a method according to claim
 12. 15. The compoundaccording to claim 14 that modulates hDAGKβ activity.
 16. A method oftreatment or prophylaxis of a disorder that is responsive to modulationof hDAGK activity in a human patient, which comprises administering tosaid patient an effective amount of a compound according to either claim14 or claim
 15. 17. A method of treatment or prophylaxis of a disorderthat is responsive to modulation of hDAGK activity in a human patient,which comprises administering to said patient an effective amount of amodulator of hDAGK activity.
 18. The method according to either claim 16or claim 17 wherein the disorder is a mood disorder, epilepsy, aneurodegenerative disorder, anxiety, schizofrenia, migraine, drugdependence, stroke, Alzheimer's dementia or Parkinson's disease.
 19. Themethod according to any one of claims 16 to 18 wherein the disorder isresponsive to modulation of hDAGKβ.
 20. Use of a compound according toeither claim 14 or claim 15 in a method of formulating a medicament fortreatment or prophylaxis of a disorder that is responsive to modulationof hDAGK activity in a human patient.
 21. Use of a modulator of hDAGKactivity in a method of formulating a medicament for treatment orprophylaxis of a disorder that is responsive to modulation of hDAGKactivity in a human patient.
 22. The use according to either claim 20 orclaim 21 wherein the disorder is a mood disorder, epilepsy,neurodegenerative disorder, anxiety, schizofrenia, migraine, drugdependence, stroke, Alzheimer's dementia, Parkinson's disease.
 23. Theuse according to any one of claims 20 to 22 wherein the disorder isresponsive to modulation of hDAGKβ.
 24. A method of producing an hDAGKβprotein or a variant thereof comprising introducing into an appropriatecell line a suitable vector comprising a nucleotide sequence encodingfor an hDAGKβ protein or a variant thereof, under conditions suitablefor obtaining expression of the hDAGKβ protein or variant.